Cytokines are key mediators of innate and adaptive immunity. Many cytokines have been used for therapeutic purposes in patients with advanced cancer, but their administration is typically associated with severe toxicity, hampering dose escalation to therapeutically active regimens and their development as anticancer drugs. To overcome these problems, the use of ‘immunocytokines’ (i.e. cytokines fused to antibodies or antibody fragments) has been proposed, with the aim to concentrate the immune-system stimulating activity at the site of disease while sparing normal tissues1-5.
The heterodimeric cytokine interleukin-12 (IL-12) is a key mediator of innate and cellular immunity with potent antitumour and antimetastatic activity6-8. It consists of a p35 and a p40 subunit covalently linked by a disulphide bridge.
Secretion of the isolated p35 subunit has never been detected; in contrast, the cells that produce the biologically active IL-12 heterodimer secrete p40 in free form in a 10-100-fold excess over the IL-12 heterodimer; depending on the stimulus9. A biological function of free p40 has never been observed and its physiological significance is still debated. Disulphide linked homodimers of p40 are produced in the mouse; murine p40 homodimers, in contrast to the free p40, have the ability to block IL-12 functions in vitro and in vivo10. The existence of human p40 homodimers has been demonstrated up to now only in p40 transfected cell lines and the physiological relevance of human p40 homodimers is still debated11,12.
IL-12 acts primarily on T and NK cells. The most important functions of IL-12 are the priming of the T helper 1 (Th1) immune responses and IFN-γ secretion by NK cells13.
IL-12 generates the Th1 response in three modalities: (i) it promotes the differentiation of naïve T cells, during initial encounter with an antigen, into a population of Th1-cells capable of producing large amounts of IFN-γ following activation14, (ii) it serves as a costimulus required for maximum secretion of IFN-γ by differentiated Th1 cells responding to a specific antigen15, and (iii) it stimulates the development of IFN-γ producing Th1 cells from populations of resting memory T cells interacting with an antigen to which they have been previously exposed16.
IL-12 strongly inhibits neo-vascularisation and IFN-γ seems to play a critical role as a mediator of the anti-angiogenic effects of IL-1217. Interferon gamma-induced protein 10 (IP-10) is known to be a potent inhibitor of angiogenesis18,19.
As with many other cytokines, however, the administration of recombinant human IL-12 is associated with severe toxicity, hampering its development as an anticancer drug. Clinical trials in patients with cancer have revealed promising therapeutic activities, but have also shown that recombinant human IL-12 is extremely toxic to humans, with a maximal tolerated dose of 0.5 μg/kg of body weight20,21.
The toxic side effects of toxins, particularly cytokines such as such as IL-12 have made it difficult to administer an effective dose and to reach high concentrations at the site of a tumour.
Previously, researchers have attempted to overcome these drawbacks by targeting delivery of IL-12 to the tumour environment and in particular to tumour blood vessels (tumour vascular targeting). Tumour vascular targeting aims at disrupting the tumour vasculature, reducing blood flow to deprive the tumour of oxygen and nutrients, causing tumour cell death.
A targeted delivery of IL-12 to the tumour environment is expected to increase the therapeutic index of the cytokine. The concentration of cytokines, and in particular IL-12, at the level of tumour blood vessels is an attractive therapeutic strategy for a number of reasons.
First, the tumour neovasculature is more accessible to intravenously administered therapeutic agents than are tumour cells, which helps avoid problems associated with the interstitial hypertension of solid tumours22.
Second, angiogenesis is characteristic of most aggressive solid tumours23. Angiogenesis describes the growth of new blood vessels from existing blood vessels. Tumours can induce angiogenesis through secretion of various growth factors (e.g. Vascular Endothelial Growth Factor). Tumour angiogenesis allows tumours to grow beyond a few millimeters in diameter and is also a prerequisite for tumour metastasis. New blood vessels formed as the result of angiogenesis form the neovasculature of the tumour or the tumour metastases. Targeting IL-12 to the neovasculature should allow the immunotherapy of a variety of different tumour types.
Third, IL-12 shows an anti-angiogenic activity conferred by its downstream mediator, IP-1017,24.
The alternatively spliced extra domains A (ED-A) and B (ED-B) of fibronectin and the A1 domain of tenascin-C represent three of the best-characterised markers of angiogenesis and have been reported to be expressed around the neo-vasculature and in the stroma of virtually all types of aggressive solid tumours. Furthermore, even non-solid cancers, such as leukaemia, may be amenable to treatment by targeting antigens of the neovasculature. WO2011/015333 described treating leukaemia, including acute myeloid leukaemia, by targeting the bone marrow neovasculature.
Three human monoclonal antibodies specific to these targets have been developed and moved to clinical trials: L19 (specific to ED-B)25, F8 (specific to ED-A)26 and F16 (specific to the A1 domain of tenascin-C)27.
In addition, several antibody derivatives, based on the modification of L19, F8 or F16 with cytokines or iodine radionuclides, are currently being investigated in Phase I and Phase II clinical trials in patients with cancer and with rheumatoid arthritis28,29. These biopharmaceuticals are called L19-124I, L19-131I, L19-IL2, L19-TNF, F8-IL10, F16-124I, F16-131I, F16-IL2, indicating the modular nature of these derivatives, in which the antibody moiety is used to deliver a payload at the site of disease.
In WO2008/120101 an I125-labelled F8 diabody was shown to selectively target I125 to tumours in mice.
An F8-IL2 diabody conjugate has been shown to reduce tumour burden in mice (WO2008/120101, WO2010/078945).
Researchers have attempted to improve targeting of IL-12 to the vasculature using antibody-IL-12 conjugates. Halin et al. sequentially fused the p40 and p35 domains of the heterodimeric IL-12 using a (Ser4Gly)3 linker and appended at the N-terminal end of the antibody fragment scFv(L19). This immunocytokine showed an increased therapeutic activity of IL12; however, only a modest tumour targeting was observed30.
Gafner et a(successfully cloned and tested a heterodimeric fusion protein in which the disulphide-linked p35 and p40 subunits were fused to scFv(L19)31 to produce the fusion protein p40-scFv(L19)/scFv(L19)-p35 (see also WO2006/119897). This heterodimeric fusion protein showed an excellent tumour-targeting performance in biodistribution studies and enhanced therapeutic activity compared to the Halin format.